Microorganisms often respond to the input of xenobiotics into the environment by evolving mechanisms to use them as sources of nutrients and energy for their growth. As the structure of the herbicides based on a s-triazine ring differ from naturally occurring compounds (Esser et al. 1975), microorganisms slowly evolved enzymes and pathways capable of degrading them. The amidohydrolase superfamily comprises a remarkable set of enzymes that catalyze the hydrolysis of a wide range of substrates bearing amide or ester functional groups at carbon and phosphorus centers. In all cases, the nucleophilic water molecule is activated through complexation with a mononuclear or binuclear metal center. In the mononuclear metal centers, the substrate is activated by a proton transfer from the active site, and the water is activated by metal ligation and general base catalysis. The metal centers are perched at the C-terminal end of the beta-barrel core within a (beta alpha) 8 structural domain. One prominent example is the Atrazine chlorohydrolase (AtzA) an Fe(II)-dependent homohexamer (Seffernick et al. 2002; Wackett et al. 2002a) catalyzing the hydrolytic dechlorination of atrazine, a herbicide, yielding the nonherbicidal product 2-hydroxyatrazine (de Souza et al. 1996; Seffernick et al. 2002; Sadowsky and Wackett 2000). The closest known relative of AtzA is melamine deaminase (TriA from Pseudomonas sp. strain NRRL B-12227; 98% sequence identity). Despite their high sequence similarity, AtzA and TriA are catalytically distinct; TriA is a deaminase with a dechlorinase activity several orders below its physiological deaminase activity, while AtzA a dechlorinase with no detectable deaminase activity. Previous work has shown that three of the nine amino acids that differ between the two proteins (S331C; N328D; and F84I AtzA) are largely responsible for the differences in catalytic specificity.
The present invention provides new methods to increase herbicide tolerance in plants by the introduction of bacterial genes encoding target proteins that biodegrade the herbicide, in particular cellulose biosynthesis inhibitors named azines. The bacterial enzyme TriA was engineered in a form to remain or increase the amidohydrolase activity and to expand the enzyme pocket towards a more bulky substrate acceptance. The inventors of the present invention have surprisingly found that over-expression of wildtype or mutant melamine deaminase TriA forms confers in plants tolerance/resistance to particular classes of herbicides as compared to the non-transformed and/or non-mutagenized plants or plant cells, respectively. More specifically, the inventors of the present invention have found that TriA expression confers tolerance/resistance to azines.
The problem of the present invention can be seen as to the provision of novel traits by identifying target polypeptides, the manipulation of which makes plants tolerant to herbicides.
Three main strategies are available for making plants tolerant to herbicides, i.e. (1) detoxifying the herbicide with an enzyme which transforms the herbicide, or its active metabolite, into non-toxic products, such as, for example, the enzymes for tolerance to bromoxynil or to basta (EP242236, EP337899); (2) mutating the target enzyme into a functional enzyme which is less sensitive to the herbicide, or to its active metabolite, such as, for example, the enzymes for tolerance to glyphosate (EP293356, Padgette S. R. et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme so as to produce quantities of the target enzyme in the plant which are sufficient in relation to the herbicide, in view of the kinetic constants of this enzyme, so as to have enough of the functional enzyme available despite the presence of its inhibitor.
The problem is solved by the subject-matter of the present invention.